EGFRvIII is a tumor specific, ligand-independent, constitutively active variant of the epidermal growth factor receptor. Our pilot results demonstrated that 70 percent (103/147) of invasive breast cancer express EGFRvIII. 86 percent (43/50) of ErbB-2 positive primary breast tumors were co-expressed with EGFRvIII. Even more striking was that 100 percent (4/4) of ErbB-2 positive metastatic lymph nodes co-overexpressed EGFRvIII. Transfection and expression of EGFRvIII in human breast cancer cell line (MCF-7) led to increase in colony formation and significantly enhanced tumorigenicity of MCF-7 cells in athymic nude mice. ErbB-2 phosphorylation was enhanced in EGFRvIII transfected MCF-7 cells. These results indicated that EGFRvIII could activate ErbB-2 kinase activity. In addition, despite the encouraging results from the advanced-disease trials with an anti-ErbB-2 antibody, Herceptin (rhuMAb HER2 or trastuzumab) clearly does not cure all tumors that overexpress ErbB-2. Based on these findings, we hypothesize that co-expression of EGFRvIII with ErbB-2 plays a critical role in human breast cancer progression. Co-expression of EGFRvIII with ErbB2 in breast cancer progression may influence Herceptin efficacy. Our ultimate goal is to develop diagnostic, prognostic, and predictive markers for Herceptin efficacy and responsiveness, as well as mechanistic rationale for novel therapeutic strategies. To achieve these goals, we propose studies with the following Specific Aims. 1): To elucidate the cross-talk between EGFRvIII with ErbB-2 and the biological effects of EGF-like ligands in a model system (32D cells): this involves generation of stable cell lines, which ectopically expresses EGFRvIII in pairwise combination with ErbB-2 in 32D cells. The biological effects of receptor cross-talk between the EGFRvIII and ErbB-2 receptor as well as ligand-induced proliferation will be investigated. 2): To determine the biological functional effects of co-expression of EGFRvIII in ErbB-2 overexpressing breast cancer cells: Non-tumorigenic, overexpressing ErbB-2 breast cancer cell lines will be transfected with EGFRvIII. The biological and biochemical effects on cell proliferation, tumorigenicity and metastatic potential in these transfectants will be defined in vitro and in vivo. 3): To determine whether co-expression of EGFRvIII modulates the Herceptin effect in ErbB-2 overexpressing cells: The impact of co-expression of EGFRvIII with ErbB-2 in Herceptin responsiveness will be evaluated by monitoring the inhibition of cell proliferation and tumorigenicity in three model systems (32D, N1H3T3 and EGFRvIII transfected overexpressing ErbB-2 breast cancer cell lines). 4): To validate the results from Aim 1 through Aim 3 in samples from breast cancer patients: Immunohistochemical analysis will be conducted to verify the frequency of EGFRvIII and co-expression with ErbB-2 with large cohorts of primary breast cancer specimens, metastatic lymph node specimens, as well as the clinical samples from patients in clinical trials with Herceptin. The correlation of their expression with other clinicopathological prognostic indicators in human breast cancer will be determined. These studies will provide us with new, important information relating the biological significance and functions of EGFRvIII co-expression of ErbB-2 in human breast cancer. Through these studies, we will gain valuable insights and identify potential markers, which may be good predictors for the efficacy of trastuzumab in treating ErbB-2-positive breast cancer patients. The results of the proposed studies may ultimately point to new directions of molecular therapies of breast cancer.